Detection of Helicobacter Pylori CagA Gene and Its Association with Endoscopic Appearance in Balinese Dyspepsia Patients

Background: Helicobacter pylori (H. pylori) infection causes various abnormalities in the stomach. Only particular strain can cause severe problems in the stomach. CagA is a microbial virulent factor which is associated with more severe stomach problems, such as: peptic ulcer and stomach cancer. We would like to know the prevalence of CagA in Balinese population, and the association of H. Pylori CagA status with the severity of endoscopic appearance in dyspepsia patients.Method: Study design being used was analytic cross sectional study, involving 71 dyspepsia patients who underwent upper gastrointestinal endoscopic examination in Surya Husada Hospital and Balimed Hospital in June-December 2013. Sample was chosen in consecutive manner. Later, polymerase chain reaction (PCR) examinations of the stomach mucous biopsy tissue to determine H. pylori infection status and CagA status were performed. Further, Chi square test was used to identify the difference in proportion of H. pylori and CagA between mild and severe endoscopic appearance.Results: In this study, we found that the prevalence of H. pylori infection was 22.5% using PCR examination. Prevalence of CagA positive in H. pylori positive was 62.5%. There was significant association between status of H. Pylori infection and severity of endoscopic appearance (p = 0.038; OR= 2.67; 95% CI = 1.18-6.05). Status of CagA in H. pylori infected patients was not associated with the severity of endoscopic appearance. Additionally, there was significant association between patients' age and severity of endoscopic appearance.Conclusion: The prevalence of CagA in H. pylori positive was 62.5%. H. pylori infection was associated with severity of endoscopic appearance and CagA status in H. pylori infected patients was not associated with severity of endoscopic appearance.


INTRODUCTION
Recently, Helicobacter pylori (H. pylori) infection is recognised as a health problem across the globe. H. pylori colonisation in human's stomach occurs in up to 50% of worldwide population and 70% of these are found in developing countries. 1,2 The prevalence of infection in South East Asia is also high, where in Thailand the prevalence was 54.1-76.1%. 3 Meanwhile, seroepidemiology in Indonesia showed the prevalence of H. pylori infection is approximately 36-46.1%. 4 In Bali, we have performed several studies in different populations. A study in a rural-based community which was performed in Tabanan showed prevalence of 43%, while another study in Denpasar revealed a prevalence of 41.2%. 5 H. pylori infection is the most common cause of chronic gastritis, and is closely related to peptic ulcer, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer. [1][2][3]6,7 In 1994, International Agency for Research on Cancer (IARC) and World Health Organization (WHO) classified H. pylori as a definite carcinogenic group. 8 The development of gastric carcinoma involves several factors, including H. pylori virulence level, host genetic characteristic, environment factor and or host-microbe specific interaction. H. pylori heterogeneity has been studied to identify the possibility of virulence factor because most H. pylori infection did not cause cancer. 9 CagA Helicobacter pylori is the first bacterial oncoprotein which has been identified for its association with human cancer. 10 Patient who is infected by strain containing CagA, has an increased risk to develop gastric cancer compared to those infected by strain which did not contain this oncoprotein. 11 CagA is the main virulence factor of H. pylori. Recent publication on the role of virulence factor to abnormalities happening in gastric mucosa in Indonesia which was performed in 5 big islands, Sumatra, Java, Borneo, Sulawesi, and Papua concluded that most infection that happened were infection of CagA strain East Asia type, but with low virulence. 12 Currently, there is no publication on the role of CagA virulence factor to the severity of gastric mucosa abnormality in Balinese population.
Based on the aforementioned background, we established formulation of research problems as follows: what is the prevalence of CagA H. pylori in dyspepsia patients and how is the association of H. pylori infection and CagA with endoscopic appearance in Balinese dyspepsia patients. This study aimed to identify the prevalence of H. pylori infection using PCR method in Balinese dyspepsia patients, to identify the prevalence of CagA in Balinese dyspepsia patients, to reveal the association of H. pylori status with endoscopic appearance, and to discover the association of CagA H. pylori status with endoscopic appearance.

METHOD
This was a study using analytic cross sectional design to identify the association between CagA H. pylori status and the endoscopic appearance of dyspepsia patients. It was conducted in Endoscopy Unit Surya Husada Hospital and Balimed Bali Hospital and Mataram Biomedik Laboratory. This study was performed for 6 months from June-December 2013. Target population of this study was all Balinese dyspepsia patients in Denpasar. Accessible population of this study was dyspepsia patients in Balimed Hospital and Surya Husada Hospital who underwent upper gastrointestinal Detection of Helicobacter pylori CagA gene and Its Association with Endoscopic Appearance in Balinese Dyspepsia Patients endoscopy examination. Samples of this study were dyspepsia patients in Balimed Hospital and Surya Husada Hospital who fulfilled inclusion and exclusion criteria. Inclusion criteria included patients with dyspepsia complaints, agreed to underwent endoscopy and gastric biopsy, aged 18 years old or more, and Balinese. Patients who refused to participate in the study, those with unstable hemodynamic condition, those who consumed PPI drugs in the last 2 weeks, NSAID drugs or antibiotics in the last 1 month.
Sample size was determined by using formula quoted from Madiyono et al, with zα 1.96, prevalence of Hp CagA+: 80%, with desired level of absolute precision of 10%; thus, based on the calculation using those numbers above, we found that the minimal number of sample needed was 69 samples. 13 Before this study was conducted, the researcher asked for permission to the Director of Balimed Hospital and Surya Husada Hospital, Denpasar, and established cooperation with nurses in the endoscopy unit. Study sample was collected through consecutive sampling. Dyspepsia patients who came to undergo endoscopic examination were explained about this study, if agreed they signed an informed consent. Patients performed preparation for endoscopic examination including fasting 6 hours before the procedure being performed. Endoscopic examination was performed by a gastroenterologist. During endoscopy, biopsy of patients' gastric mucosa was performed, one from the corpus area and one from the antrum area. Results of endoscopic examination were presented in the form of pictures with description of endoscopic appearance being found. Gastric mucosal biopsy was then preserved in buffer media, for further PCR examination in Mataram Biomedik Laboratory. Collected data was analysed using computer software.

RESULTS
In this study, 71 samples who fulfilled inclusion and exclusion criteria were included. Thirty-eight individuals (53.5%) were males and the other 33 were (46.5%) females. Patients who participated in this study were between 21-70 years old. Samples' age was normally distributed (Kolmogorov Smirnov; p = 0.2). Patients who were included in this study were all Balinese living in Bali; endoscopic examination was performed in 2 private hospitals in Denpasar. PCR examination of H. pylori was performed in Mataram Biomedik Laboratory. Complete sample characteristics were presented in Table 1. In this study, we found 16 (22.5%) patients from 71 samples were positive for PCR UreC examination, and found 10 (62.5%) individuals with positive CagA from 16 patients with positive PCR results. To perform analysis of study results, endoscopic appearance (EA) was classified into 2 main group: mild abnormality and severe abnormality. Superficial antrum gastritis 43 (60.6%) and superficial pangastritis 12 (16.9%) were classified into mild abnormality, while erosive gastritis 7 (9.9%), gastric ulcer 4 (5.6%), duodenal ulcer 3 (4.2%) and gastric polyp 2 (2.8%) were classified into severe abnormality. To define which variables influence the endoscopic appearance, we performed analysis to age, sex, and education level variables of the samples. The analysis results could be seen in Table 2.  pylori infected patients and severity of the patients' endoscopic appearance.

DISCUSSION
In this study, we obtained that the prevalence of H. pylori infection using PCR method was 22.5%. Meanwhile, using IgG Anti H. pylori examination, we found that the prevalence was 18.3%. These results were quite low compared to the previously performed studies using different methods of examination. Generally, prevalence of H. pylori reaches up to 80% in developing countries in middle-aged population, while in developed countries the prevalence was 20-50%. In children, the incidence of H. pylori infection was quite low, but will increase up to 20% in the age of 40 and 50% in the age of 60. 15 The result of this study was similar to the study performed by Darya in 2009 which found that the prevalence of H. pylori infection in gastritis patients in Sanglah Hospital was 18.8% using immunochromatography method. 16 Target population in both studies was similar, which was dyspepsia patients who visited each hospitals for treatment. Meanwhile, other studies had different target population, which was general population who had dyspepsia complaints got higher results. A study in rural area in Tabanan obtained a prevalence of 43%, while another study in Denpasar found a prevalence of 41.2%. 5 Latest study in Indonesia attained a prevalence of 33.6%. 12 PCR is a very good diagnostic tool to detect the presence of H. pylori infection. In the study performed by Fonseca et al, it was found that the sensitivity and specificity to detect H. pylori using ureA PCR was 93.3 and 95.8%, meanwhile if culture is used, the result showed 100% sensitivity and specificity. 17 Additionally, compared to H. pylori examination using immunohistochemical analysis and campylobacter like   From the table above, we could see that CagA positive was performed in 5 from 9 PCR (+) in mild endoscopic appearance (superficial gastritis), and 5 from 7 PCR (+) in severe EA (3 from 4 PCR (+) in erosive gastritis, 1 from 2 PCR (+) in gastric ulcer and 1 from gastric polyp). Relationship between CagA status and severity of endoscopic appearance could be seen in Table 5. From that cross table, Chi-square test was performed. Because there was more than 1 cell having expected value < than 5, then the Chi-square Detection of Helicobacter pylori CagA gene and Its Association with Endoscopic Appearance in Balinese Dyspepsia Patients organism (CLO) test, PCR examination can identify more samples. This is proven by study performed by Weiss et al which found 23 H. pylori positive samples using PCR but negative using immunohistochemical analysis or even using CLO test. 18 A study in Netherland showed that in order to detect H. pylori, PCR examination has the highest sensitivity of 99.4%, followed by histological examination (92.2%), culture (89.5%) and CLO test (89.0%). For their specificities, these 3 examinations had specificity of more than 98%. This study also showed that the positive results obtained from CLO test (p = 0.001) and culture (p = 0.031) was very influenced by bacterial density.
However, this is not the case in examination using PCR method (p = 0.130). Therefore, effect of mistakes during sample collection for PCR examination was very small, because it did not require a large amount of sample for examination using PCR method. 19 In this study, we observed that age was associated with the severity of endoscopic appearance. The older patients showed more severe endoscopic appearances. In accordance to the results of previous studies. Prevalence of normal gastric mucosa was said to decrease in line with the addition of age, either with or without the presence of H. pylori infection. 20 In older patients, there will be a decreased capacity of defensive factors in gastric mucosa to maintain gastric mucosal integrity. It may relate to the decrease in the production of mucous and bicarbonate. In healthy old individuals, it is also found that there is a decrease in prostaglandin concentration compared to younger individuals; this is known to be the result of significant decrease of cyclooxygenase enzyme activity. Therefore, it is easier to experience abnormality compared to younger age. 21 H. pylori strain which contains CagA gene is said to cause infection with worse clinical symptoms. Although CagA is not H. pylori infection determinant, identifying the presence of this gene may help to determine the prognosis and association of infection with the occurring disease. 17,19 In this study, we found 62.5% patients with positive PCR had CagA gene. This result was in accordance to the previous study in which this gene was reported to be found in approximately 60-70 % from H. pylori isolates in Europe. 22 Other study found a prevalence of 68.3%, while a study conducted by Fonseca reported a prevalence of 53%, where 6 (75%) patients suffered from erosive gastritis and 2 (25%) patients suffered from superficial gastritis. 17,23 Another study even indicated a lower prevalence of 40.8%. 24 In this study, we found significant association between clinical manifestations and CagA status. However, they found that the prevalence of peptic ulcer was more common in CagA EPIYA-ABCC/ABCCC strain compared to CagA EPIYA-ABC strain. 24 This showed that microbial virulence was not only determined by the presence of CagA gene, but also by the role of EPIYA motif in the CagA gene.
Recent study found that the virulence level of H. pylori was determined by genotype of EPIYA motif in CagA. Patients with EPIYA Western type (ABC) motif had milder clinical appearance compared to EPIYA East Asian type (ABD) because EPIYA ABD motif would produce more IL-8 from gastric epithelial cells compared to EPIYA ABC motif. 27 In addition to CagA, there are several microbial virulence factors which need to be considered that influence the severity of gastric abnormality, such as VacA, OipA, IceA, BabA. VacA type s1/m1 is the most active type which has the potential to cause more severe abnormality. 28 Infection of gastric mucosa by H. pylori is marked by the production of proinflammatory cytokine, particularly interleukin 8 (IL-8), which is a potent neutrophil activation chemokine. Cag PAI is involved in the induction of gastric IL-8 production. A study showed that CagA was involved in the induction of I Ketut Mariadi, I Dewa Nyoman Wibawa, Ida Bagus Nyoman Putra Dwija IL-8 depend on the strain. IL-8 level induced by CagA East Asian type was higher compared to those induced by CagA Western type. 29 This probably underscores more severe clinical manifestation to happen due to infection of H. pylori strain CagA East Asian type.
A Study performed in 5 big islands in Indonesia revealed that the prevalence of CagA was 97.7% from patients with positive H. pylori, with dominant of EPIYA East Asian motif, but clinical symptoms which appear mostly were mild. This was thought to be caused by EPIYA gene East Asian type which was found in Indonesia had deletion in 6 bp, thus it had different type to the EPIYA gene East Asian type. 12 Bali Island which is located in Indonesia, probably had CagA strain similar to this study, thus also had CagA strain with EPIYA East Asian motif with mild clinical appearances.

CONCLUSION
In this study, we found prevalence of CagA in H. pylori infection was 62.5%. H. pylori status was associated with severity of endoscopic appearance. Meanwhile, CagA status from H. pylori was not associated with severity of endoscopic appearance. Further study is needed to identify the EPIYA motif from H. pylori strain with positive CagA to determine the virulence of H. Pylori strain.